R E V I E W Open Access
Molecular genetics of human primary microcephaly: an overview
Muhammad Faheem1, Muhammad Imran Naseer2,3*, Mahmood Rasool2,3, Adeel G Chaudhary2,
Taha A Kumosani1,4, Asad Muhammad Ilyas1, Peter Natesan Pushparaj2,3, Farid Ahmed2,3, Hussain A Algahtani5, Mohammad H Al-Qahtani2,3, Hasan Saleh Jamal6
From2nd International Genomic Medicine Conference (IGMC 2013) Jeddah, Kingdom of Saudi Arabia. 24-27 November 2013
Abstract
Autosomal recessive primary microcephaly (MCPH) is a neurodevelopmental disorder that is characterised by microcephaly present at birth and non-progressive mental retardation. Microcephaly is the outcome of a smaller but architecturally normal brain; the cerebral cortex exhibits a significant decrease in size. MCPH is a neurogenic mitotic disorder, though affected patients demonstrate normal neuronal migration, neuronal apoptosis and neural function. Twelve MCPH loci (MCPH1-MCPH12) have been mapped to date from various populations around the world and contain the following genes:Microcephalin,WDR62,CDK5RAP2, CASC5, ASPM, CENPJ, STIL, CEP135,CEP152, ZNF335, PHC1andCDK6. It is predicted that MCPH gene mutations may lead to the disease phenotype due to a disturbed mitotic spindle orientation, premature chromosomal condensation, signalling response as a result of damaged DNA, microtubule dynamics, transcriptional control or a few other hidden centrosomal mechanisms that can regulate the number of neurons produced by neuronal precursor cells. Additional findings have further elucidated the microcephaly aetiology and pathophysiology, which has informed the clinical management of families suffering from MCPH. The provision of molecular diagnosis and genetic counselling may help to decrease the frequency of this disorder.
Introduction
In this review article, we discuss the clinical manifesta- tions of autosomal recessive primary microcephaly (MCPH), its incidence, and molecular genetics, including a comprehensive review of the twelve known mapped loci (MCPH1-MCPH12). Further, we review the corre- sponding genes and the proteins encoded by these genes, their possible role in the developing brain and reported mutations of these genes. In addition, the potential for these genes to perform various cognitive roles during human brain evolutionary processes is discussed.
Clinical features of MCPH
Microcephaly is characterised by a reduced occipitofrontal circumference (OFC) of the head that is at least 4 standard
deviations (SD) and is caused by congenital insufficiency during fetal brain development, which chiefly affects the cerebral cortex. The current clinical definition of MCPH is as follows: A: a head circumference (HC) of at least 4 SD below the age- and sex-matched means [1]; B: intellectual impairment that is not related to a neurological finding, such as spasticity or progressive cognitive decline [1]; and C: most MCPH patients are of a normal height, weight, and appearance and have normal chromosome analysis and brain scan results [2,3]. The only exception of this is observed in patients with mutations in the microcephalin gene who have a clinically defined short stature and abnormal chromosome analysis results [4-6]. Microce- phaly in MCPH is primary, is evident by the 32nd week of gestation, can be observed at the time of birth and is non-progressive [7,8]. HC or OFC is the most common diagnostic tool used for MCPH. Based on the findings of several studies conducted on more than 200 families worldwide, it is believed that the specific physical
* Correspondence: [email protected]
2Center of Excellence in Genomic Medicine Research, King Abdulaziz University, KSA
Full list of author information is available at the end of the article
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appearance of patients linked to each MCPH locus is indistinguishable [9-12]. Additionally, the chromosomal analysis for most MCPH patients was reportedly normal [9]. However, recent studies supported by modern neuro- diagnostic modalities, such as computed tomography (CT) scan and magnetic resonance imaging (MRI), have signifi- cantly increased our knowledge regarding the discernible clinical presentations of MCPH and have expanded the definition of MCPH based on phenotype [7,13,14].
Molecular Genetics of MCPH MCPH Incidence
Primary microcephaly is present at birth and is a static developmental anomaly, whereas secondary microce- phaly develops postnatally and is a progressive neurode- generative condition. Primary microcephaly is very rare, and the rate of incidence is estimated to be 1 in 30,000 in Japan [15] and 1 in 250,000 in Holland [16]. The inci- dence is increased up to 1 in 10,000 in regions where marriages between cousins are common [1,6]. The birth incidence of primary microcephaly differs from 1.3 to 150/100,000, depending on the population type and consanguineous populations [17].
MCPH Inheritance
MCPH is inherited in an autosomal recessive pattern in which both copies of the gene in each cell have mutations.
Individuals with MCPH inherit one copy of the mutated gene from each parent, but the parents typically do not demonstrate any signs or symptoms of the condition [18].
Genes in MCPH
MCPH is a neurogenic mitotic disorder that does not occur due to abnormal neuronal migration or neural apoptosis.
Rather, it develops because all of the identified MCPH genes are expressed in the neuroepithelium; the phenotypic features and brain scans of MCPH patients reveal a smaller than average brain size. Brain size during birth is deter- mined by both the proliferation rate and cell death rate during neurogenesis. The majority of neurons originate from neural progenitors, which are the predominant cells of the neuroepithelium (NE) lining the brain ventricles [1].
The precise mitotic activity of neuronal progenitor cells was first described in fly neuroblasts:symmetricalcell divi- sions with mitotic spindles oriented in the neuroepithelium plane produce two neuronal progenitor cells, whereas asymmetriccell division occurs when the mitotic spindles are perpendicularly oriented toward the neuroepithelium and results in one postmitotic neuron and one progenitor cell [1]. Because the timing of these divisions during devel- opment is critical for the final number of neurons, any change in its regulation could lead to cortical disorders, such as microcephaly [19]. MCPH exhibits genetic hetero- geneity, and to date, twelve loci (MCPH1-MCPH12) have
been associated with this condition [20]. A comprehensive review is outlined below of the twelve MCPH genes that carry mutations, their possible functions in causing the disease (Table 1), various mutations in people of different ethnic backgrounds (Table 2) and animal model studies (Table 3) of the MCPH genes.
1- Microcephalin (MCPH1)
MCPH1 encodes the BRCT (BRCA1 C-terminus) domain-containing protein microcephalin; microcepha- lin contains three BRCT domains, which are evolutiona- rily conserved phospho-peptide-interacting amino acid tandem repeat domains and are significant in cell cycle control. MCPH1 is characterised by a congenital decrease in brain size, especially in the cerebral cortex.
The microcephalin gene is localised on chromosome 8p23, is composed of 14 exons and 835 amino acids, and has a genome size of 241,905 bp, with three already known isoforms and an 8,032 bp open reading frame [17,21]. The first documented MCPH1 gene mutation was observed in Pakistani families [21]; additionally, a large deletion mutation covering the first 6 exons of the microcephalin gene was reported in an Iranian family that exhibited autosomal recessive mental retardation and mild microcephaly [22]. An observed missense mutation in MCPH1 demonstrated a less severe cellular phenotype and mild microcephaly [5,7]. Later, a muta- tion in the same gene was identified in patients with microcephaly disorder, a misregulated chromosomal condensation termed premature chromosome condensa- tion (PCC) syndrome and short stature [23,24]. A higher expression of this gene was observed in the fetal mouse brain during neurogenesis, especially in ganglionic emi- nences and lateral ventricles [21]. It was revealed that both MCPH1 and the PCC syndrome are allelic disor- ders originating from mutations within the same gene [4]. This protein is also involved in DNA damage- induced cellular responses, chromosomal condensation and during G2/M checkpoint arrest through maintain- ing the inhibitory cyclin-dependent kinase phosphoryla- tion [6]. Further studies revealed that the consequences of MCPH1 deficiency in Drosophila include a slow mitotic entry, PCC, mitotic entrance with non-replicated DNA, non-coordinated centrosome, genome instability and a detached centrosome. Additionally, mitotic arrest was observed in Drosophila cells with deficient MCPH1 [25,26]. It was also observed that because of the influ- ence on premature neurogenic formation, the progeni- tors with reduced MCPH1 increased the production of early born neurons, which comprise the deep layers (IV–VI), and decreased the late-born neurons, which produce the thinner outer cortex layer (II–III). However, neuronal migration is not affected due to MCPH1 dele- tion [27]. The successful generation of a mouse model
for MCPH1-related primary microcephaly revealed misregulated mitotic chromosome condensation due to defective MCPH1 function and a reduced skull size [28,29].
2-WDR62(MCPH2)
TheWDR62gene is localised inside the MCPH2 locus at chromosome 19q13.12 and has 32 exons and a genomic size of 50,230 bp [30].WDR62is a WD40 repeat-contain- ing protein expressed in neuronal precursors and post- mitotic neurons in the developing brain and is localised in the spindle poles of dividing cells. Phenotypic variations of WDR62advocate its key role in numerous aspects of cere- bral cortical development [13]; it has been linked with
severe brain malformations. A previous study revealed five homozygousWDR62mutations, including deletions and premature terminations, in patients of Turkish origin suf- fering from severe brain malformations and microcephaly [30]. A 7.5 Mb locus on chromosome 19q13.12 composed of 148 genes was recognised in two families via genome- wide linkage analysis. Additionally, high throughput sequencing of associated genes in each family produced more than 4,000 DNA variants harbouring deleterious changes involving a single geneWDR62. Additionally, six mutations were observed in theWDR62gene through tar- geted high throughput sequence analysis in six families suffering from a congenital microcephaly syndrome and diverse defects in cerebral cortical architecture [13].
Table 1 Summary of known MCPH genes, their location and role.
Locus Gene Cytoband Cellular location Role/effect on brain development References
MCPH1 Microcephalin 8p23.1 Nucleus Involved in chromosomal condensation, reduced MCPH1
enhances the production of early born neurons, which comprise deep layers (IV–VI), and reduces the late-born neurons, which
produce the thinner outer cortex layer (II–III).
[6,27]
MCPH2 WDR62 19q13.12 Nucleus/Centrosomes/
Neuronal precursors/Post- mitotic neurons
Cerebral cortical development, proliferation and migration of neuronal precursors, mutation inWDR62affects its role in proliferating and migrating neural precursors and causes severe
brain malformations.
[13,30]
MCPH3 CDK5RAP2 9q33.2 Centrosome Regulates microtubule function, mutation inCDK5RAP2reduces the progenitor pool/decreases the number of neurons and
reduces cell survival.
[35,36,38,88]
MCPH4 CASC5 15q15.1 Kinetochore Vital for the spindle checkpoint of the mitotic cycle,CASC5 underscores the role of kinetochore integrity in the proper
volumetric development of the human brain.
[40,41]
MCPH5 ASPM 1q31.3 Pericentrosomal Orientation of mitotic spindles during embryonic neurogenesis, ASPMmutations can decrease the size of the brain by influencing
the orientation of the mitotic spindle.
[57,54,14]
MCPH6 CENPJ 13q12.12 Centrosome/
neuroepithelium of the frontal cortex
Controls centriole length/microtubule function, its deletion causes an increased incidence of multiple spindle poles, apoptosis and
mitosis arrest
[63,64]
MCPH7 STIL 1p33 Pericentrosomal Apoptosis regulator/cell cycle progression, its mutation in zebrafish causes an embryonic lethal defect, andSTILknockout
mice (Sil-/-) exhibit numerous developmental abnormalities/
decreased size/defective midline neural tube.
[72,69,68]
MCPH8 CEP135 4q12 Centrosome Maintains organisation/structure of the centrosome,CEP135
knockdown showed decreased growth rate/disorganised microtubules.
[72,70,20]
MCPH9 CEP152 15q21.1 Centrosome Centriole duplication/shape to cell/polarity/motility, conversion of glutamine into proline disturbs potential coiled-coiled protein
domain/reduced head size.
[76,77]
MCPH10 ZNF335 20q13.12 Nucleus Progenitor cell division/differentiation, mutatedZNF335gene causes degeneration of neurons, knockdown ofZNF335caused a small brain size with an absent cortex/disrupted proliferation and
differentiation of neuronal cells.
[78]
MCPH11 PHC1 12p13.31 Nucleus Regulates cell cycle,PHC1mutation highlights the role of
chromatin remodelling in the pathogenesis of Primary Microcephaly.
[80]
MCPH12 CDK6 7q21.11 Cytoplasmic/nuclear Controls cell cycle/organises microtubules,CDK6mutation affects apical neuronal precursor cells proliferation/reduces progenitor
pool/decreases neuronal production/primary microcephaly.
[83]
Abbreviations. MCPH: Autosomal recessive primary microcephaly; MCPH1: Microcephalin;WDR62:WD repeat-containing protein 62;CDK5RAP2: Cyclin dependent kinase-5 regulatory subunit associated protein; CASC5:Cancer susceptibility candidate-5;ASPM:Abnormal spindle like primary microcephaly;CENPJ: Centromere- associated protein J;STIL:SCL/TAL1interrupting locus;CEP135:Centrosomal protein 135;CEP152:Centrosomal protein 152; ZNF335: Zinc Finger Protein 335;PHC1:
Polyhomeotic-like protein 1;CDK6: Cyclin-dependent kinase 6.
Table 2 Number of reported mutations in MCPH genes in families of different ethnic backgrounds worldwide.
MCPH genes
Reported mutations
Types of mutations Ethnicity (families) References
Missense/
nonsense
Splicing Deletions &
insertions
Complex rearrangements
MCPH1 24 8 1 14 1 Iranian (8), Caucasian (1), Pakistani (13) [5,22,51,86,89-91]
WDR62 28 14 1 13 0 Iranian (3), Turkish (11), Mexican (1), Arab (1),
Pakistan (9)
[7,13,30,86,91]
CDKRAP2 6 3 2 1 0 Pakistani (2), Somalian (1) [7,32,34,36,92]
CASC5 1 1 0 0 0 Moroccan (3) [43]
ASPM 104 48 7 48 1 Iranian (13), Arab (12), Indian (8), European (22),
African (5), Pakistani (60), many sporadic cases.
[7,14,50,51,86,91,93]
CENPJ 5 2 1 2 0 Iranian (5), Pakistani (6) [32,60,61,86,91]
STIL 4 2 1 1 0 Iranian (2), Indian (3) [68,86,87]
CEP135 1 0 0 1 0 Pakistani (1) [20]
CEP152 9 5 2 2 0 Pakistani (5), Canadian (3) [77,91,94]
ZNF335 2 1 1 0 0 Arab Israeli (1) [78]
PHC1 1 1 0 0 0 Saudi Arabian (1) [80]
CDK6 1 1 0 0 0 Pakistani (1) [83]
Abbreviations. MCPH: Autosomal recessive primary microcephaly; MCPH1: Microcephalin;WDR62:WD repeat-containing protein 62;CDK5RAP2:Cyclin dependent kinase-5 regulatory subunit associated protein; CASC5:Cancer susceptibility candidate-5;ASPM:Abnormal spindle like primary microcephaly;CENPJ: Centromere- associated protein J;STIL:SCL/TAL1interrupting locus;CEP135:Centrosomal protein 135;CEP152:Centrosomal protein 152; ZNF335: Zinc Finger Protein 335;PHC1:
Polyhomeotic-like protein 1;CDK6: Cyclin-dependent kinase 6.
Table 3 MCPH animal models.
MCPH gene
Animal orthologs of human gene
Animal model References
MCPH1 mcph1-/-(Drosophila/
mice)
Premature condensation of chromosome, non-coordinated centrosome, genome instability, detached centrosome, changes in cell cycle progression and defects in DNA damage repair,
misregulated mitotic chromosome condensation.
[25,26,29]
CDK5RAP2 Cnn-/-(Drosophila) Centrosome dysfunction causes connection loss between centrosomes and pericentriolar matrix.
Only subtle defects of asymmetric divisions; the sizes of the observed brains were normal.
[39]
ASPM asp-/-(Drosophila) Aspm-/-(mice)
The Drosophilaaspgene is vital for both the organisation and binding together of microtubules at the spindle poles and to focus the poles of the mitotic spindles during mitosis and meiosis.
Two mutant mouse lines showed that mutations inASPMdecreased the brain size in mice.
[56-58]
CENPJ Sas-4-/-(Drosophila,c.
elegans)
Centrioles are lost due to damage of theCENPJorthologuedsas-4 in Drosophila; in contrast, the knockout flies can live until adulthood but have weak coordination and lower viability. The morphological development of mutant flies is normal without cilia or flagella, but they die in
early life.
[65,66]
STIL STIL Sil-/-(mice) STILknockout mice (Sil-/-) exhibit numerous developmental abnormalities in embryonic days E7.5-8.5 and die after E10.5, the phenotype of the mutant mice is characterised by decreased brain size, restricted development, defective midline neural tube, abnormal development, and
enhanced apoptosis; the observed mutants were embryonically lethal.
[71]
CEP135 Bld10-/- (Chlamydomonas)
In Chlamydomonas,CEP135orthologBld10pmutants (bld10) demonstrated abnormal interphase microtubules and mitotic spindles, defects during cell division and a significant decrease in the
growth rate.
[73]
CEP152 Orthologous (Drosophila asterless;asl)
CEP152is the putative mammalian orthologue ofDrosophila asterless, mutations in which affect mitosis in the fly.
[77]
ZNF335 Orthologue (mice;
ZNF335)
Knockdown ofZNF335caused a small brain size with an absent cortex and disrupted the proliferation and differentiation of neuronal cells. It was observed that knockdown of theZNF335
ortholog in mice (ZFP335) resulted in early embryonic lethality at day E7.5. Conditional knockdown ofZNF335in mouse cortical cells resulted in a small brain with a fundamentally
absent cortex with deficient cortical neurons.
[78]
Abbreviations. MCPH1: Microcephalin;CDK5RAP2:Cyclin dependent kinase-5 regulatory subunit associated protein; ASPM:Abnormal spindle like primary microcephaly;CENPJ: Centromere-associated protein J;STIL: SCL/TAL1interrupting locus;CEP135:Centrosomal protein 135;CEP152:Centrosomal protein 152;
ZNF335: Zinc Finger Protein 335.
Published observations from multiple groups have shown thatWDR62is mainly nuclear in its location and lacks any apparent association with the centrosomes. In contrast to this, other findings have shown that theWDR62expres- sion is similar toASPM, which is also a MCPH protein focused at the spindle poles in neuronal precursor cells [30]. Another study further confirmed that disturbance of the centrosome integrity and spindle defects are the key players in developing MCPH2 microcephaly [31]. In HeLa cells,WDR62accumulates in the centrosome or nucleus in a cell cycle-dependent manner; siRNA knockdown of WDR62in cortical progenitors decreased their prolifera- tion, caused defective spindle orientation, and reduced the centrosome integrity, displacement from the spindle poles and late mitotic development [31].
3-CDK5RAP2(MCPH3)
Homozygous mutations of cyclin-dependent kinase 5 regu- latory subunit-associated protein-2 (CDK5RAP2) have been recognised as the basis of MCPH3 [32]. This protein is composed of 38 exons encoding 1,893 amino acids. Three CDK5RAP2mutations (two in Pakistani families; one in a Somalian patient) have been described; 1: a nonsense muta- tion in exon 4 (246T>A); 2: the A to G transition in intron 26 (4005-15A>G) produces a novel splice acceptor site, frame shift and a premature stop codon; and 3: a nonsense mutation in exon 8 (700G>T). All of these mutations result in a truncated protein withCDK5RAP2functional loss [32-34].CDK5RAP2is strongly linked with the centrosome, Golgi apparatus, and microtubules and is chiefly present in the neural progenitors of the ventricular and sub-ventricu- lar zones of an immature brain; it has also been observed in glial cells and early born neurons and is progressively down regulated as the brain matures [35,36].CDK5RAP2is also essential in the regulation of the spindle checkpoint; its loss causes chromosomal missegregation and decreased spindle checkpoint protein expression through binding with their promoters and transcriptional regulation in HeLa cells [37].
A model of the microcephaly phenotype due to a mutation inCDK5RAP2causes a premature transition from sym- metric to asymmetric neuronal progenitor cell division with a consequent reduction of the progenitor pool, decreased neurons and reduced cell survival [35,38]. Centrosome dys- function, which is known as centrosome‘rocketing’, was observed within the cells because of the connection loss between the centrosomes and pericentriolar matrix in the humanCDK5RAP2ortholog‘centrosomin’-deficient Droso- phila embryos (cnn-/-). Only subtle asymmetric division defects were observed in‘centrosomin’-deficient Drosophila embryos (cnn-/-), and the brain sizes were normal [39].
4-CASC5(MCPH4)
CASC5is a 265 KDa large protein that belongs to the con- served KMN protein network and permits chromosomal
kinetochore docking to the microtubule. Its location adja- cent to the kinetochore remains constant from G2 until late anaphase; proper docking is very important for suffi- cient sister chromatid segregation at anaphase and is vital for the spindle checkpoint of the mitotic cycle [40,41]. A CASC5mutation on the MCPH4 locus with an LOD score of > 6 in MCPH patients from three consanguineous families has been reported. This mutation enhanced the skipping of exon 18, produced a frame shift and premature stop codon in exon 19, followed by truncation of the pre- dicted protein. Mutation 6125G>A inCASC5covers the amino acids from 1,981 to 2,108, which interact with ZWINT-1, whereas theCASC5C-terminus is important for binding with NSL1 and the MIS12 complex in a down- stream portion of the mutation. Therefore, the loss of exon 18 and the resulting frame shift and truncation lead to a functional loss ofCASC5[40,42]. This protein is also upregulated in the ventricular zone of the human fetal brain and interacts with BUBR1, ZWINT-1 and MIS12 through the N-terminal domain, which can be truncated due to mutation [43]. A transcriptome analysis through RNA sequencing of the germinal zone of the human fetal neocortex at 13-16 gestational weeks revealed a higher expression ofCASC5in the ventricle zone compared the sub ventricle zone and cortical plate; this finding further confirmed the effect of mutatedCASC5on proliferating cells in the ventricle zone that cause microcephaly [43,44].
Further analysis byCASC5siRNA knockdown in HeLa cells revealed chromosomal misalignment and the prema- ture entry of the cells into mitosis; this defect causes improper symmetrical and asymmetrical division, which results in inefficient proliferation, inappropriate neuronal number and abnormal brain size [45].
5-ASPM(MCPH5)
Abnormal spindle-like primary microcephaly (ASPM), which is homologous to Drosophila melanogaster, is impor- tant for the normal functioning of the mitotic spindle in embryonic neuroblasts harbouring MCPH5 locus [14]. It consists of 62,567 bp with a 10,906 bp ORF, 28 exons and 3,477 amino acids, with a putative microtubule-interacting domain at the N-terminus [46,47], calponin homology domain, 81 isoleucine glutamine motifs acting as a calmo- dulin binding domain [14,48,49], and a C-terminus without any identified domains [50]. Studies have revealed four novel mutations of this gene in 33 families from Pakistan [51]. ASPM gene mutations truncate the protein and include deletion, single base pair change, duplication and variation in the intronic region. Additionally, substantial numbers of the observedASPMmutations are compound heterozygous [10,52]. In neurogenesis, this gene is mainly expressed in the cerebral cortical ventricular zone and pro- liferation zones of the medial and lateral ganglion [14].
Another study showed the localisation of this protein at the
spindles, and its knockdown by siRNA revealed reduced proliferative symmetric division in the mouse developing neocortex [53]. Studies have shown that because of neuro- sphere differentiation,ASPMis downregulated in mice;
additionally, neurosphere proliferation and self-renewal was decreased as a result ofASPMknockdown [54]. Mor- pholino-mediated knockdown of ASPM in zebrafish resulted in a significant reduction in head size [55]. Droso- philaaspgene mutants were the first to be known as hav- ing a mini-brain; thisaspgene is known to be vital for both the organisation and binding of microtubules at the spindle poles and its ability to focus the poles of mitotic spindles during mitosis and meiosis [56,57]. Additionally, two mutant mouse lines were generated from gene trap cells for the study ofASPMfunction in the development of the cerebral cortex. The results showed that mutations inASPMdecreased the size of brain in mice [58]. It is therefore concluded thatASPMmutations can decrease the size of the brain by influencing the orientation of the mitotic spindle and can decrease the neuronal cells by affecting the asymmetrical to symmetrical cell division ratios [54,57].
6-CENPJ(MCPH6)
Mutations in the centromere-associated protein J (CENPJ) gene harboured in the MCPH6 locus on human chromo- some 13q12.2 have been reported to cause primary micro- cephaly [32]. This gene is composed of 40,672 bp with a 5,187 bp ORF [46] and 17 exons, which encode a protein of 1,338 amino acids; it contains 5 coiled-coiled domains, many protein phosphorylation sites, 21 nonamer G-box repeats in the C-terminal domain and, C-terminal to that, contains a leucine zipper motif [32,59]. In total, four mutations were identified in theCENPJgene: one muta- tion in one Brazilian family, [32] and one mutation in two Pakistani families [32]. Later, another Pakistani family showed the deletion of four consecutive nucleotides (TCAG) in the 11thexon of theCENPJgene, which causes a frame shift and a premature termination codon 19 bp downstream in the same exon that is predicted to add six amino acids downstream of the mutation [60]. The fourth mutation is a splicing mutation in theCENPJgene that was identified in Seckel syndrome patients [61]. This pro- tein is mainly expressed in the neuroepithelium of the frontal cortex at the beginning of neurogenesis [32].
Depletion of this protein disturbs the centrosome integrity, and cells deficient inCENPJ become arrested during mitosis, with multipolar spindles [62]. Anin vitroanalysis showed that this protein can impede microtubule nuclea- tion and depolymerisation, predicting a role forCENPJin directing the centrosomal assembly of microtubules [63].
Prior studies have also shown thatCENPJ deletion by siRNA causes the enhancement of multiple spindle poles, apoptosis and mitosis arrest [64]. Centrioles are lost
due to damage of theCENPJorthologuedsas-4 in Dro- sophila; in contrast, the knockout flies can live until adulthood but have weak coordination and lower viabi- lity [65]. The morphological development of mutant flies is normal without cilia or flagella, but they die in early life [66].Sas-4 is a centriole protein controlling centrosome organisation inC. elegans, and its involve- ment has been confirmed in the duplication of centro- some fluorescence recovery after photo bleaching (FRAP).CENPJregulates the centriole length through- out biogenesis, with possible involvement in the accu- rate recruitment of centriolar microtubules [67].
7-STIL(MCPH7)
TheSCL/TAL1interrupting locus (STIL) gene was identi- fied at the seventh locus for MCPH, spanning 63,018 bp with a 5,225 bp ORF, 20 exons and 1,287 residues in a full-length cytosolic protein of 150 kDa; it also contains a putative nuclear localisation signal and C-terminal domain [17,68]. Three mutations causing protein truncation were observed in members of five Indian families in an 8.39 Mb region of chromosome 1p33-p32.3 [68]. The expression of this gene is observed throughout the cytosol but mainly in the peri-nuclear region, with an essential role in mitotic entry (G2-M phase), apoptosis regulation and centrosome function [68].STILis a primary response gene that is ubi- quitously expressed in proliferating cells and in early embryonic development [69,70]. Studies of zebrafish have revealed thatSTILhas an important role in the duplication of centrosomes and in the organisation of mitotic spindles.
STILmutation in zebrafish causes an embryonic lethal defect [69], and STIL knockout mice (Sil-/-) exhibit numerous developmental abnormalities at embryonic day E7.5-8.5 and die after E10.5. The phenotype of the mutant mice is described as a decreased size, restricted develop- ment, defective midline neural tube, abnormal develop- ment, enhanced apoptosis, reduced proliferation and abnormal expression of numerous essential genes, includ- ing lefty-2 and nodal. Additionally, the observed mutants were embryonically lethal [71].
8-CEP135(MCPH8)
Centrosomal protein (CEP135) is encoded by theCEP135 gene, is composed of 26 exons and 1,140 amino acids and is a conservedahelical protein observed throughout the cell cycle at the centrosome. As a centrosomal component, this protein gives more strength to the centrosomes. A truncated mutation inCEP135was identified in a Pakis- tani family at locus 4q12 that caused microcephaly. A deletion mutation 970delC changes glutamine to serine at position 324, followed by the premature termination of a codon. Considering the mutation’s position and the affected protein; the truncated mutation is definitely not compatible with the normal function of CEP135[20].
Studies by electron microscopy revealed thatCEP135is associated with pericentriolar material, an electron-dense material around the centrioles. In interphase, the mitotic spindles are disorganised due to an RNAi-mediated decreased amount ofCEP135in the cells, which suggests thatCEP135maintains the organisation and structure of the centrosomes and microtubules [72]. In Chlamydomo- nas,CEP135orthologbld10pmutants (bld10) demon- strated abnormal interphase microtubules and mitotic spindles, defects during cell division and a significant decrease in the growth rate [73]; additionally, CEP135 knockdown inCHO cells showed a decreased growth rate [72]. The C-terminal domain ofCEP135contains an interacting site for its two binding partners, the 50 kDa dynactin complex subunit (p50) and C-Nap1. Due to the truncation ofCEP135, both p50 and C-Nap1 are released, promoting the decomposition of centrosomes and the pre- mature splitting of the centrosomes, respectively [74,75].
9-CEP152(MCPH9)
CEP152is an important 152 kDa centrosome protein that organises the microtubules of animal cells and has an essential role in giving shape to the cell, polarity, motility and cell division [76]. TheCEP152gene is localised to the MCPH4 locus at chromosome 15q21.1; recently, microce- phaly due to a mutation in theCEP152gene was desig- nated as MCPH9 [43,77]. HumanCEP152is an ortholog of the Drosophila asterless (asl) gene, contains 72,835 bp and 1,710 amino acids encoding a 152 kDa protein [77]. Three families with one microcephalic child each were identified from a subpopulation of eastern Canadian. A homozygosity analysis of two of the families by genome-wide dense SNP genotyping revealed the locus 15q21.1 chromosome. Exon sequencing of the candidate genes identified a non-con- served amino acid conversion into a highly conserved CEP152residue [77]. One mutation converts glutamine into proline, which is anticipated to be pathogenic and can disturb a potential coiled-coiled protein domain. The other mutation causes protein truncation by eliminating a por- tion predicted to be a coding region from the C-terminal domain [77]. A greater head size reduction was observed in compound heterozygous females compared with missense homozygous females. The truncated protein causes a non- sense-mediated decay of the mutated transcript; studies have also revealed during a functional assay to observe the subcellular localisation that the wild typeCEP152-GFP fusion protein can be identified ing-tubulin-co structures [77]. MutantCEP152flagged with GFP failed to colocalize with theg-tubulin, which further confirmed the pathogeni- city due to this mutation [77].
10-ZNF335(MCPH10)
The ZNF335 gene encodes a component of the verte- brate-specific, trithorax H3K4-methylation chromatin
remodelling complex, which regulates neuronal gene expression and cell fate [78]. MCPH10 was identified due to a mutation in theZNF335gene in affected mem- bers of an Arab Israeli family. This mutation was recog- nised in a homozygous 3332G-A transition in exon 20 of the ZNF335 gene and resulted in an Arg1111 to His (R1111H) substitution at a conserved residue in the 13th zinc-finger domain. This mutation was identified at the final position of a splice donor site that disturbed normal splicing, leading to an abnormally large tran- script that included introns 19 and 20 with a predicted premature termination [78]. Patient cells also showed severely reduced levels of ZNF335 protein, but a few residual transcripts were identified. Cellular studies revealed that ZNF335binds to the chromatin remodel- ling complex, including H3K4 methyl transferases that can regulate the expression of specific genes in various pathways; the complex is analogous to the TrxG (trithorax) complex in Drosophila. Mutations in nBAF, one of the neural-specific chromatin regulatory com- plexes, affected proliferation, revealing that it is related to microcephaly [79]. Insufficiency of theZNF335 gene causes the degeneration of neurons, which makes it different and much more severe compared with other microcephaly syndromes that are well linked to postnatal survival.In vitroandin vivomouse models showed that knockdown ofZNF335caused a small brain size with an absent cortex and disrupted the proliferation and differen- tiation of neuronal cells [78]. It was observed that knock- down of theZNF335ortholog in mice (ZFP335) resulted in early embryonic lethality at day E7.5. Conditional knockdown ofZNF335in mouse cortical cells resulted in a small brain with a fundamentally absent cortex with defi- cient cortical neurons. Knockdown ofZNF335in mouse neuronal progenitor cells caused reduced proliferation and self-renewal due to premature cell cycle exit. Similar find- ings were observed in anin vivoembryonic mouse model usingin uteroelectroporation to targetZNF335in cortical progenitor cells.ZNF335depletion resulted in abnormal neuronal cell orientation and radial glia with disorganised dendritic outgrowth. [78].
11-PHC1(MCPH11)
Primary microcephaly-11 (MCPH11) is caused by homo- zygous mutation in the PHC1 gene on chromosome 12p13. A 12-year-old girl and her 6-year-old brother, who were born to related Saudi parents, suffered from primary microcephaly (-5.8 and -4.3 SD, respectively) and low nor- mal cognitive function. The brain MRIs were normal, except for a small brain size. The sister and brother also had short stature (-3.6 SD and -2.3 SD, respectively) [80].
The mutation, which was found by homozygosity mapping combined with exome sequencing, segregated with the disorder and was not found in the dbSNP, Exome Variant
Server or 1000 Genomes database or in 199 Saudi exomes or 554 Saudi control individuals [80]. A microarray analysis showed that various genes with a role in regulating the cell cycle are dysregulated in the patient cells [80]. Cells with a PHC1mutation also enhanced the geminin expression, which was recapitulated in RNAi experiments in control and patient cells in whichPHC1was ectopically expressed.
Because geminin has a recognised role in controlling the cell cycle [81,82], PM pathogenesis could be partly due to abnormalities in the cell cycle that are the result of accu- mulated geminin. The patient cells also showed an increase in DNA damage and defective DNA repair in response to irradiation and abnormal cell cycle activity consistent with reduced proliferative activity compared with controls.
These defects were associated with abnormalities in chro- matin regulation and could be rescued in patient cells by the overexpression of wild typePHC1[80].
12-CDK6(MCPH12)
The cyclin-dependent kinase 6 (CDK6) protein controls the cell cycle and differentiation of different cell types.
Prior studies showed a genetic defect through a novel MCPH locus on the long arm of chromosome 7 (MCPH12). This mutated gene causes disorganisation of the microtubules and spindles, distorted nuclei, and super- numerary centrosomes. Furthermore, this role was con- firmed by knockdown cells and cells expressing theCDK6 mutant allele, whereas the decreased proliferation observed in the patient and knockdown cells reinforced its already described role in controlling the cell cycle [83].
CDK6mutation also affects apical neuronal precursor cell proliferation and produces an imbalance between sym- metric and asymmetric cell division, which results in a progenitor pool reduction that could be the reason of decreased neuronal production and, finally primary micro- cephaly [83]. A study of a consanguineous eight-genera- tion Pakistani family with ten microcephalic children showed a new MCPH locus at HSA 7q21.11-q21.3 [83].
Most related candidate genes in this region showed a homozygous single nucleotide substitution, 589G>A, in CDK6, which encodes cyclin-dependent kinase 6. This mutation leads to an abnormal mitotic morphology and has an effect on cellular localisation because patient pri- mary fibroblasts failed in recruitingCDK6to the centro- some during the mitosis process [84]. This finding was confirmed by another study that showed a lower prolifera- tion capacity of CDK6 patient primary fibroblasts and knockdown cells; CDK6 also has a significant role in dif- ferentiating various cell types [85].
Mutation spectrum of MCPH Genes in Middle Eastern and Saudi populations
A total of approximately forty-three families have been reported with mutations in MCPH gene from the Middle
Eastern and, especially, Saudi Arabian region: twenty-eight from Iran, fourteen from Arab countries, three from Mor- occo and one from Saudi Arabia [22,43,80,86,87] . More specifically, eight Iranian families have been reported with mutations in the MCPH1 gene, thirteen with mutations in theASPMgene, five with mutations in theCENJPgene and two with mutations in the STILgene [22,86,87].
CASC5 gene mutation have been found in MCPH patients from three consanguineous Moroccan families with a homozygous missense mutation 6125G>A that leads to an amino acid substitution Met2041Ile; this substitution is predicted to inactivate an exonic splicing enhancer (ESE), leading to an abnormal transcript lacking exon 18 [43]. A total of thirteen Arab families have been reported with mutations in theASPMandZNF335genes. A homozy- gous 2974C>T mutation in thePHC1gene on chromo- some 12p13 was reported in a 12-year-old girl and her 6- year-old brother born to related Saudi parents who were suffering from primary microcephaly [80].
Conclusions
Cognitive capabilities are reduced with a substantial decrease in brain size in MCPH patients, whose brain sizes decrease to one third compared with its original size with a substantial cognitive decline. There are twelve known genes that cause this neurodevelopmental disorder, and recent evidence suggests that MCPH is possibly a primary disorder of neurogenic mitosis. With the progress of MCPH gene identification, prenatal diag- nosis (detects a disorder recurrence), postnatal diagnosis (differentiates the disorder from various differential diagnosis) and carrier testing (for consanguineous families in which the disease is known to occur) are increasingly available for patients. The successful crea- tion of mammalian models for MCPH has enabled researchers to further add to the knowledge regarding the aetiology and pathophysiology of MCPH. Better gen- otyping, neuro-physiological and neuroimaging testing, together with the creation of genetically homogeneous groups of patients, would benefit the identification of exact genotype phenotype correlations. Mutational screening in different MCPH families from the Middle East and especially Saudi Arabia would help in genetic counselling and prenatal diagnosis for MCPH and would eventually enable us to reduce the incidence of MCPH in a highly consanguineous population. Addi- tionally, these studies have increased our understanding of the control of neuronal production by neural stem cells and how this has been modulated by evolution to control the brain sizes of different species.
Abbreviations
MCPH: Autosomal recessive primary microcephaly; OFC: Occipitofrontal circumference; SD: Standard deviations; HC: Head circumference; PCC:
Premature chromosome condensation; MRI: Magnetic Resonance Imaging;
WDR62:WD repeat-containing protein 62;CDK5RAP2: Cyclin dependent kinase-5 regulatory subunit associated protein-2;CASC5:Cancer susceptibility candidate-5;ASPM:Abnormal spindle like primary microcephaly;CENPJ:
Centromere-associated protein J; FRAP: Fluorescence recovery after photo bleaching;CEP:Centrosomal protein;ZNF335: Zinc Finger Protein 335;PHC1:
Polyhomeotic-like protein 1;CDK6:Cyclin-dependent kinase 6.
Competing interests
The authors declare that they have no conflicts of interest.
Authors’contributions
MF, MIN and PNP wrote the manuscript. MR, AGC, TAK, MHA, AMI, HSJ and FA edited the final version. All authors read and approved the final version of the manuscript.
Acknowledgments
The authors gratefully acknowledge the King Abdulaziz City for Science and Technology (KACST) Large Grant No# (APR-34-13) and KACST Project Code:
Project No.8-MED120-3 for providing the funds and for its continual generous support during the course of this important project, KSA. MR is supported by KACST strategic project code“12-MED3078-03; PNP is supported by KACST strategic project“12-BIO2719-03 and 12-BIO2267-03; FA supported by KACST strategic project code“09-BIO693-03; HSJ is supported by KACST project code“8-MED 120-3”.
Declarations
This article has been published as part ofBMC Medical GenomicsVolume 8 Supplement 1, 2015: Selected articles from the 2nd International Genomic Medical Conference (IGMC 2013): Medical Genomics. The full contents of the supplement are available online at http://www.biomedcentral.com/
bmcmedgenomics/supplements/8/S1
Authors’details
1Department of Biochemistry, Faculty of Science, King Abdulaziz University, KSA.2Center of Excellence in Genomic Medicine Research, King Abdulaziz University, KSA.3KACST Technology Innovation Center in Personalized Medicine, King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia.4King Fahd Medical Research Center, King Abdulaziz University, KSA.5King Saud bin Abdulaziz University for Health Sciences, KSA.6Department of Obstetrics and Gynecology, King Abdulaziz University Hospital, Jeddah, Kingdom of Saudi Arabia.
Published: 15 January 2015
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